Efficient Synthesis, Structural Characterization, Antibacterial Assessment, ADME-Tox Analysis, Molecular Docking and Molecular Dynamics Simulations of New Functionalized Isoxazoles

This work describes the synthesis, characterization, and in vitro and in silico evaluation of the biological activity of new functionalized isoxazole derivatives. The structures of all new compounds were analyzed by IR and NMR spectroscopy. The structures of 4c and 4f were further confirmed by single crystal X-ray and their compositions unambiguously determined by mass spectrometry (MS). The antibacterial effect of the isoxazoles was assessed in vitro against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus bacterial strains. Isoxazole 4a showed significant activity against E. coli and B. subtilis compared to the reference antibiotic drugs while 4d and 4f also exhibited some antibacterial effects. The molecular docking results indicate that the synthesized compounds exhibit strong interactions with the target proteins. Specifically, 4a displayed a better affinity for E. coli, S. aureus, and B. subtilis in comparison to the reference drugs. The molecular dynamics simulations performed on 4a strongly support the stability of the ligand–receptor complex when interacting with the active sites of proteins from E. coli, S. aureus, and B. subtilis. Lastly, the results of the Absorption, Distribution, Metabolism, Excretion and Toxicity Analysis (ADME-Tox) reveal that the molecules have promising pharmacokinetic properties, suggesting favorable druglike properties and potential therapeutic agents.


Introduction
Pathogenic microorganisms like bacteria are responsible for many serious infections, threatening well-being and even leading to large-scale health crises [1,2].Combating these bacterial infections has become a serious and persistent concern for the global scientific community, due to the increased resistance of microorganisms caused by random genetic mutations [3,4].Moreover, the widespread and inappropriate use of existing antibacterial drugs contributes to the proliferation of microorganisms that are resistant to antibacterial treatments [5,6].This multi-drug resistance poses a major risk, leading to high morbidity and mortality rates [7,8].Therefore, there is an urgent need to discover more effective therapies and develop new powerful antibacterial drugs, capable of effectively targeting a wide range of microorganisms.
Heterocyclic compounds indeed offer a rich reservoir of molecular structures with diverse biological activities, making them valuable in drug discovery to combat infectious diseases [9].Medicinal chemists continue to focus on the modification of heterocyclic molecules to develop new therapeutic agents with improved efficacy and safety profiles [10,11].Additionally, heterocyclic chemistry in recent years has undergone considerable development due to the diverse biological activities exhibited by many heterocyclic compounds [12,13].The distinctive features of heterocyclic motifs lie in their structures in saturated, partially saturated, or aromatic heterocyclic compounds, as well as in functional groups [14,15].They are the active ingredient of many families of both natural and synthetic products effective in therapeutic, agrochemical [16,17], industrial [18], and other fields.

Introduction
Pathogenic microorganisms like bacteria are responsible for many serious infections, threatening well-being and even leading to large-scale health crises [1,2].Combating these bacterial infections has become a serious and persistent concern for the global scientific community, due to the increased resistance of microorganisms caused by random genetic mutations [3,4].Moreover, the widespread and inappropriate use of existing antibacterial drugs contributes to the proliferation of microorganisms that are resistant to antibacterial treatments [5,6].This multi-drug resistance poses a major risk, leading to high morbidity and mortality rates [7,8].Therefore, there is an urgent need to discover more effective therapies and develop new powerful antibacterial drugs, capable of effectively targeting a wide range of microorganisms.
Heterocyclic compounds indeed offer a rich reservoir of molecular structures with diverse biological activities, making them valuable in drug discovery to combat infectious diseases [9].Medicinal chemists continue to focus on the modification of heterocyclic molecules to develop new therapeutic agents with improved efficacy and safety profiles [10,11].Additionally, heterocyclic chemistry in recent years has undergone considerable development due to the diverse biological activities exhibited by many heterocyclic compounds [12,13].The distinctive features of heterocyclic motifs lie in their structures in saturated, partially saturated, or aromatic heterocyclic compounds, as well as in functional groups [14,15].They are the active ingredient of many families of both natural and synthetic products effective in therapeutic, agrochemical [16,17], industrial [18], and other fields.
Isoxazoles are an attractive type of heterocyclic compound for medicinal research due to their broad spectrum of biological activity.These include ones having antihistaminic [19], antifungal [20,21], antimicrobial, [22,23], antiviral [24,25], anti-inflammatory [26,27], antioxidant [22,28], and anticancer [29,30] activities as well as ones used as herbicides [31], fungicides [32], insecticides [33], and anticorrosive [34][35][36] coatings, among others.Indeed, there are several drugs currently on the market which incorporate the isoxazole moiety as the main pharmacophore (Figure 1).Also, isoxazoles are precursors to many bioactive molecules, including amino acids, amino-alcohols, and amino esters [23,24].Chemoinformatics has indeed emerged as a crucial tool in various scientific disciplines, including chemistry, biology, and materials science [35,36].These techniques have been widely used to discover new candidates because they offer a powerful alternative for predicting and interpreting complex experimental data.In particular, in silico molecular docking studies are of great importance in drug design, as they can predict the likely interactions between potential drug molecules (ligands) and their target proteins (enzymes or receptors) [37,38].Moreover, molecular dynamics simulation analysis allows researchers to track the behavior of complexes formed between ligands and target proteins under in silico physiological conditions, exploring and evaluating their stability over time [39,40].These computational simulations provide valuable insights that complement experimental methods and enhance the efficiency of the drug development process.
Building upon the observed beneficial effects demonstrated by the above-mentioned type of isoxazole compounds and as a continuation of our research on the synthesis and biological evaluation of new heterocyclic systems [41][42][43], we report here the functionalization of new isoxazole systems synthesized from (Z)-2-benzylidenebenzofuran-3(2H)-one (aurone) through the benzoyloxy and acetoxy groups, as well as an assessment of their antibacterial activity using both in vitro and in silico approaches.

Syntheses of Functionalized Isoxazoles 4 and 5
Initially, the precursors 3(a-c) were synthesized by a 1,3-dipolar cycloaddition (1, reaction between the dipolarophile (Z)-2-benzylidenebenzofuran-3(2H)-one 1 and 1,3dipoles of the arylnitriloxide type (Scheme 1).These were generated in situ from chlorinated aldoximes 2(a-c) and triethylamine in chloroform at ambient temperature in the presence of 1 to prevent the dimerization of the dipole into furoxane.This reaction results in the formation of isoxazoles 3(a-c) directly instead of spiroisoxazolines [44,45].
Chemoinformatics has indeed emerged as a crucial tool in various scientific disciplines, including chemistry, biology, and materials science [35,36].These techniques have been widely used to discover new candidates because they offer a powerful alternative for predicting and interpreting complex experimental data.In particular, in silico molecular docking studies are of great importance in drug design, as they can predict the likely interactions between potential drug molecules (ligands) and their target proteins (enzymes or receptors) [37,38].Moreover, molecular dynamics simulation analysis allows researchers to track the behavior of complexes formed between ligands and target proteins under in silico physiological conditions, exploring and evaluating their stability over time [39,40].These computational simulations provide valuable insights that complement experimental methods and enhance the efficiency of the drug development process.
Building upon the observed beneficial effects demonstrated by the above-mentioned type of isoxazole compounds and as a continuation of our research on the synthesis and biological evaluation of new heterocyclic systems [41][42][43], we report here the functionalization of new isoxazole systems synthesized from (Z)-2-benzylidenebenzofuran-3(2H)one (aurone) through the benzoyloxy and acetoxy groups, as well as an assessment of their antibacterial activity using both in vitro and in silico approaches.

Scheme 1. Synthetic route of the compounds 3(a-c).
The synthesis of the functionalized isoxazoles 4(a-f) relied on the reactivity of the hydroxy group of 3(a-c) towards benzoyl chloride and acetic anhydride, respectively, forming the new isoxazole compounds with ester functional group in satisfactory yields (Scheme 2).

Scheme 1. Synthetic route of the compounds 3(a-c).
The synthesis of the functionalized isoxazoles 4(a-f) relied on the reactivity of the hydroxy group of 3(a-c) towards benzoyl chloride and acetic anhydride, respectively, forming the new isoxazole compounds with ester functional group in satisfactory yields (Scheme 2).
The structures of the functionalized isoxazole products were determined by IR, 1 H-NMR, and 13 C-NMR spectroscopies plus MS and confirmed using single-crystal X-ray diffraction for 4c and 4f.The physical and spectroscopic features of all isoxazole products are outlined in Table 1.indicating the successful formation of pure products in good yields.Specifically, the IR spectra of 4(a-f) show the disappearance of the absorption band of the alcohol function (OH) of 3(a-c) and the appearance of an absorption band around 1750 cm −1 corresponding to the C=O stretching vibration of the ester group.The 1 H-NMR spectra all indicate the disappearance of the singlet signal at 11.63 ppm assigned to the hydroxyl proton for 3(a-c).The methyl group of the ester functional group, where present, appears as a singlet at around 2.2 ppm while the 13 C-NMR spectra show, besides the peaks assigned to the aromatic carbons, a peak around 168 ppm attributable to the carbonyl C=O of the ester functional group.High-resolution mass spectroscopy showed molecular ion peaks exactly corresponding to the molecular masses of the proposed products.The structures of the functionalized isoxazole products were determined by IR NMR, and 13 C-NMR spectroscopies plus MS and confirmed using single-crystal X-ray fraction for 4c and 4f.The physical and spectroscopic features of all isoxazole prod are outlined in Table 1.indicating the successful formation of pure products in g yields.Specifically, the IR spectra of 4(a-f) show the disappearance of the absorption b of the alcohol function (OH) of 3(a-c) and the appearance of an absorption band aro 1750 cm −1 corresponding to the C=O stretching vibration of the ester group.The 1 H-N spectra all indicate the disappearance of the singlet signal at 11.63 ppm assigned to hydroxyl proton for 3(a-c).The methyl group of the ester functional group, where pre appears as a singlet at around 2.2 ppm while the 13 C-NMR spectra show, besides the p assigned to the aromatic carbons, a peak around 168 ppm attributable to the carbonyl of the ester functional group.High-resolution mass spectroscopy showed molecular peaks exactly corresponding to the molecular masses of the proposed products.a Isolated yield of products after purification.

X-ray Diffraction Data and Structural Determination of 4c and 4f
In 4c, both substituents on the C5•••C10 ring extend in the same direction, giving the molecule a "pincer-like" conformation (Figure 2).
The isoxazole ring is planar to within 0.0080(8)Å and the mean planes of the C5•••C10 and the C13•••C18 rings are inclined to the above plane by 62.39(5) and 59.44(6) • , respectively.The dihedral angle between the mean planes of the C19•••C24 and the isoxazole rings is 35.61 (4) • while that between the mean planes of the C13•••C18 and the C19•••C24 rings is 67.20(6) • .All the bond distances and interbond angles appear as expected for the formulation given.In the crystal, the C23-H23•••O2 hydrogen bonds (Table 2) form chains of molecules that extend along the c-axis direction. -----161.391609 a Isolated yield of products after purification.

X-ray Diffraction Data and Structural Determination of 4c and 4f
In 4c, both substituents on the C5•••C10 ring extend in the same directio molecule a "pincer-like" conformation (Figure 2).2) of molecules that extend along the c-axis direction.
The isoxazole ring appears planar although the slight elongation of the displacem ellipsoids for O1 and N1 normal to the plane could suggest the presence of two oppo non-planar conformers.The mean planes of the C18•••C23 and the C24•••C29 rings are clined to that of the isoxazole ring by 59.33(6) and 40.92(8)°, respectively, while the dihed angles between the mean planes of the C5•••C10 and the C12•••C17 rings and that of the is azole ring are 64.80 (7) and 55.80(7)°, respectively.All the bond distances and interbond gles appear as expected for the formulation given.In the crystal, the C28•••H28•••Cg2 in actions (Table 3) form chains of molecules extending along the c-axis direction, which paired up by inversion-related C23-H23•••Cg1 interactions (Table 3) to form ribbons (Fig 6).The ribbons pack with normal van der Waals contacts (Figure 7).clined to that of the isoxazole ring by 59.33(6) and 40.92(8)°, respectively, while the dihedral angles between the mean planes of the C5•••C10 and the C12•••C17 rings and that of the isoxazole ring are 64.80 (7) and 55.80(7)°, respectively.All the bond distances and interbond angles appear as expected for the formulation given.In the crystal, the C28•••H28•••Cg2 interactions (Table 3) form chains of molecules extending along the c-axis direction, which are paired up by inversion-related C23-H23•••Cg1 interactions (Table 3) to form ribbons (Figure 6).The ribbons pack with normal van der Waals contacts (Figure 7).

Antibacterial Screening of Isoxazoles 4(a-f)
Functionalized isoxazoles play a significant role in medicinal chemistry and biological sciences and have shown many applications in drug development for the management of a variety of life-threatening pathologies [46,47].The acetoxy 4(a-c) and the benzoyloxy 4(d-f) isoxazole derivatives were synthesized (Figure 8) and subsequently tested in vitro for their antibacterial action against Gram-positive and Gram-negative bacterial species such as Staphylococcus aureus (CECT 976), Bacillus subtilis (DSM 6633), and Escherichia coli (K12).The disk diffusion technique was used to assess bacterial susceptibility to the different isoxazoles and the results for the new isoxazoles and reference drugs are given in Table 4. d-f) isoxazole derivatives were synthesized (Figure 8) and subsequently tested in vitro for their antibacterial action against Gram-positive and Gram-negative bacterial species such as Staphylococcus aureus (CECT 976), Bacillus subtilis (DSM 6633), and Escherichia coli (K12).The disk diffusion technique was used to assess bacterial susceptibility to the different isoxazoles and the results for the new isoxazoles and reference drugs are given in Table 4.The preliminary screening results show that 4(a-f) possess variable antibacterial properties against the tested bacteria when compared to standard drugs.The most promising results against the Gram-negative bacterium Escherichia coli were shown by 4a, which carries the methyl substituent, with a zone of inhibition of 17.5 ± 1.20 mm, compared to the known antibiotic streptomycin (ZI = 24 ± 1.60).Compound 4e showed robust efficacy against Escherichia coli with an inhibition zone of 14.5 ± 0.90 mm, while 4c and 4f, both with a chlorine substituent, showed inhibition zones of 13.5 ± 1.50 and 13.25 ± 0.40 mm, respectively.The Gram-positive bacterium Bacillus subtilis was found to be less sensitive to 4(a-f).In particular, 4a exhibited good antibacterial action against Bacillus subtilis, with an inhibition zone of 14.5 ± 0.80 mm, comparable to ampicillin (ZI = 16 ± 1.30 mm) while 4c, 4d, and 4f also displayed antibacterial action against Bacillus subtilis with inhibition zones of 11 ± 1.20, 11.5 ± 1.15, and 11.25 ± 0.75 mm, respectively.For the Gram-positive bacterium strain Staphylococcus aureus, the best antibacterial activity was shown by 4f, which carries the chlorine substituent, with an inhibition zone of 15.5 ± 1.55 mm,  The preliminary screening results show that 4(a-f) possess variable antibacterial properties against the tested bacteria when compared to standard drugs.The most promising results against the Gram-negative bacterium Escherichia coli were shown by 4a, which carries the methyl substituent, with a zone of inhibition of 17.5 ± 1.20 mm, compared to the known antibiotic streptomycin (ZI = 24 ± 1.60).Compound 4e showed robust efficacy against Escherichia coli with an inhibition zone of 14.5 ± 0.90 mm, while 4c and 4f, both with a chlorine substituent, showed inhibition zones of 13.5 ± 1.50 and 13.25 ± 0.40 mm, respectively.The Gram-positive bacterium Bacillus subtilis was found to be less sensitive to 4(a-f).In particular, 4a exhibited good antibacterial action against Bacillus subtilis, with an inhibition zone of 14.5 ± 0.80 mm, comparable to ampicillin (ZI = 16 ± 1.30 mm) while 4c, 4d, and 4f also displayed antibacterial action against Bacillus subtilis with inhibition zones of 11 ± 1.20, 11.5 ± 1.15, and 11.25 ± 0.75 mm, respectively.For the Gram-positive bacterium strain Staphylococcus aureus, the best antibacterial activity was shown by 4f, which carries the chlorine substituent, with an inhibition zone of 15.5 ± 1.55 mm, compared to ampicillin (ZI = 23 ± 2.60 mm).Compounds 4d and 4e also exhibited excellent action against Staphylococcus aureus, with inhibition zones of 12 ± 1.05 and 13.75 ± 1.25 mm, respectively.To validate the outcomes of the experimentally determined biological properties, in silico studies has been conducted.

Molecular Docking Studies
The molecular docking technique is used to explore how drugs bind to the active sites of target proteins, and to identify the precise interactions involved.In this context, we carried out molecular docking simulations of 4(a-f) and proteins from different bacteria: Escherichia coli (6kzv), Staphylococcus aureus (5tw8) and Bacillus subtilis (1of0).Using these simulations, we were able to detect how these molecules bind and interact with bacterial proteins.The binding affinity results are presented in Table 5.The binding affinity observed for E. coli ranges from −9.38 to −12.84 kcal/mol, with streptomycin exhibiting the highest affinity.These high binding affinity values suggest that these molecules have considerable potential to act as potent inhibitors of the Escherichia coli protein receptor.In the case of Staphylococcus aureus, the binding affinity values range from −8.16 to −10.45 kcal/mol.Compound 4d exhibits the highest binding affinity, which surpasses that of the standard drug Ampicillin, suggesting its good potential as an antibacterial agent against Staphylococcus aureus.For Bacillus subtilis, the range of binding affinities is −8.58 to −11.17 kcal/mol, with 4f displaying the highest affinity, showing that 4f outperforms Ampicillin and indicating its promising inhibitory activity against this pathogen, thereby offering a promising alternative for addressing antibacterial resistance issues associated with this strain.Furthermore, 4b exhibits the lowest binding affinity among the tested compounds for both Staphylococcus aureus and Bacillus subtilis, which is consistent with the in vitro results.This correlation between the in silico and in vitro results reinforces the predictive value of molecular docking studies in the identification and optimization of new antibacterial agents.The energetics of the interaction of 4(a-f) associated with docking revealed a similar complexation energy for the majority of them, indicating that the molecular interactions are quite similar in all cases.To elucidate these interactions, a detailed study was carried out on 4a with the aim of identifying the essential interactions between 4a and the target proteins of different bacteria and to assess its potential as a multi-target inhibitor.The results show that 4a forms specific interactions with proteins from Escherichia coli, Staphylococcus aureus and Bacillus subtilis (shown in Figure 9) which are similar to those of the reference drugs.

Molecular Dynamics Simulation Analysis
Since 4a was identified to have the best antibacterial efficacy, the molecular docking results were supplemented by molecular dynamics (MD) simulations.Variations in the conformation and dynamics of the complexes formed between Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and 4a were quantified using the RMSD (root mean square deviation) and RMSF (root mean square fluctuation) indicators, illustrated in Figures 10 and 11, respectively.

Molecular Dynamics Simulation Analysis
Since 4a was identified to have the best antibacterial efficacy, the molecular docking results were supplemented by molecular dynamics (MD) simulations.Variations in the conformation and dynamics of the complexes formed between Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and 4a were quantified using the RMSD (root mean square deviation) and RMSF (root mean square fluctuation) indicators, illustrated in Figures 10 and  11, respectively.In the case of the Escherichia coli (6kzv) protein (Figure 10), the RMSD changes reveal a significant increase in the oscillations which rise from 0.8 to 1.6 Å in just 10 nanoseconds.This increase seems to be mainly due to the initial kinetic effect that the complexes display during the transition phase.After this phase, stabilization and equilibrium were observed for all the complexes.On the other hand, for Staphylococcus aureus (5tw8) and Bacillus subtilis (1of0), the RMSD evolutions indicate almost identical variations, with an oscillation between 1 and 1.5 Å throughout the molecular dynamics (MD) simulation.The average values (RMSD) observed for the complexes formed between 4a and the proteins of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were 1.82 Å, 1.56 Å, and 1.70 Å, respectively.The fact that these RMSD values are below 2 Å means that 4a has remarkable stability within the active sites of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis proteins.
The analysis of the RMSF trajectories provides essential information on the stability of the ligand-receptor interaction, enabling us to assess the stability, rigidity, and compactness of the protein being analyzed.A high RMSF value indicates high flexibility, meaning that the residues are less stable, while a low value indicates greater rigidity and In the case of the Escherichia coli (6kzv) protein (Figure 10), the RMSD changes reveal a significant increase in the oscillations which rise from 0.8 to 1.6 Å in just 10 nanoseconds.This increase seems to be mainly due to the initial kinetic effect that the complexes display during the transition phase.After this phase, stabilization and equilibrium were observed for all the complexes.On the other hand, for Staphylococcus aureus (5tw8) and Bacillus subtilis (1of0), the RMSD evolutions indicate almost identical variations, with an oscillation between 1 and 1.5 Å throughout the molecular dynamics (MD) simulation.The average values (RMSD) observed for the complexes formed between 4a and the proteins of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were 1.82 Å, 1.56 Å, and 1.70 Å, respectively.The fact that these RMSD values are below 2 Å means that 4a has remarkable stability within the active sites of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis proteins.
The analysis of the RMSF trajectories provides essential information on the stability of the ligand-receptor interaction, enabling us to assess the stability, rigidity, and compactness of the protein being analyzed.A high RMSF value indicates high flexibility, meaning that the residues are less stable, while a low value indicates greater rigidity and stability.For the protein from Escherichia coli (6kzv) (Figure 11), most residues display similar RMSF values, although some show notable deviations, notably at the ASP6 (3.56 Å), THR85 (2.26 Å), ALA100 (1.85 Å), GLY200 (1.76 Å), and GLY220 (3.69 Å) positions.These variations are mainly found in regions of the protein that are not directly involved in its activity, making their impact relatively low.In contrast, essential residues within the active site, such as GLU50, ASP73, ARG76, and THR165, show much more restricted movements, with RMSFs of no more than 0.9 Å, indicating the importance of the hydrogen bonds formed by these residues in stabilizing the interactions between the Escherichia coli (6kzv) protein and the ligands, thereby ensuring the greater stability of the complex.For the Staphylococcus aureus (5tw8) and Bacillus subtilis (1of0) proteins (Figure 3), the RMSF fluctuations indicate significant variations for certain residues; for example, ALA41 (2.05 Å), HIS234 (3.08 Å), GLY296 (1.84 Å), GLY330 (1.97 Å), and ASP358 (1.75 Å) in Staphylococcus aureus, and PRO217 (2.01 Å), GLY323 (2.17 Å), GLN362 (2.57Å), and PRO511 (1.91 Å) in Bacillus subtilis.These residues located outside the active sites have a limited impact on the protein's ability to bind ligands.On the other hand, residues fundamental to the function of these proteins showed low RMSF values, suggesting the existence of hydrogen bonds for stabilizing the protein-ligand complexes in Staphylococcus aureus and Bacillus subtilis.These observations confirm the results of the RMSD analysis, highlighting good structural stability in the ligand-protein complexes, mainly due to strong hydrogen bonding interactions.

ADME-Tox Analyses
The evaluation of 4a, 4b, 4c, 4d, 4e, and 4f as potential pharmaceutical agents for the treatment of bacterial infections was based on the predictions of their ADMET pharmacokinetic parameters.The results, presented in Table 6, show promising pharmacokinetic properties, suggesting their potential efficacy as therapeutic agents.The intestinal absorption rates of the compounds ranged from 97.961% to 100%, indicating the excellent absorption capacity in the human intestine, which is crucial for their oral efficacy.With regard to distribution, the predictions concerning the permeability of the blood-brain barrier (BBB) and access to the central nervous system (CNS) indicate that all the compounds analyzed show negative values for LogBB and LogPS [48].These results imply a limited distribution within these zones.This limitation is potentially beneficial in the treatment of certain bacterial infections, as it reduces the likelihood of neurological side effects by preventing drugs from reaching the CNS in large quantities.The metabolism studies showed that all the compounds acted as potential substrates and/or inhibitors of the CYP3A4 enzyme, which played a crucial role in this study [49,50].The total clearance values suggest moderate to low excretion for all compounds, suggesting that these molecules could persist in the body for a prolonged period.Finally, the predicted non-toxicity of all the compounds studied is a positive aspect that reinforces the safety profile of the ligands for further development.In summary, the ADMET prediction results offer valuable insights into the potential of the synthesized compounds as future therapeutic agents.

Summary
To conclude, this work reports the synthesis, crystallographic analysis, biological evaluation, and in silico studies of new ester-functionalized isoxazole compounds.The compounds were obtained through a series of reactions using an efficient and robust procedure starting from 2-benzylidenebenzofuran-3(2H)-one (aurone).Structural determination was achieved using IR, 1 H-NMR, and 13 C-NMR spectroscopy and mass spectrometry with further confirmation via X-ray diffraction for compounds 4c and 4f.The antibacterial screening of the synthesized ester-functionalized isoxazole compounds against three bacterial strains-Escherichia coli, Staphylococcus aureus, and Bacillus subtilis-showed that the compounds possess good antibacterial properties.Furthermore, the molecular docking studies revealed significant binding interactions between the isoxazole compounds and the bacterial target proteins of Escherichia coli (6kzv), Staphylococcus aureus (5tw8), and Bacillus subtilis (1of0).The MD simulation results indicate the remarkable stability of compound 4a within the active sites of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis proteins.Lastly, the results of the ADMET profiles of the isoxazole compounds indicate that they have good bioavailability.Taken together, the findings of this research could provide important insights to design and synthesize new ester-functionalized isoxazole compounds that are likely to be promising antibacterial agents.

Chemistry
Complete insights into the reagents and solvents required for compound synthesis and the equipment used in spectroscopic characterization and X-ray crystallographic analysis as well as the relevant synthesis procedures are clearly described in the Supplementary Material file.

Antibacterial Screening Protocol
The technique and bacterial strains used to screen the antibacterial properties of 4(a-f) are included in the Supplementary Material file.

Molecular Docking
We conducted molecular docking simulations with compounds 4(a-f) using specific target proteins from different bacteria.The proteins selected for these simulations are as follows: ➢ Escherichia coli (PDB ID: 6kzv) [51]: • Target protein: Gyrase A (DNA gyrase subunit A).

•
Biological role: Gyrase A is essential for bacterial survival as it catalyzes the negative supercoiling of DNA, which is crucial for DNA replication and transcription.Inhibiting this enzyme can prevent DNA replication, leading to cell death.
• Biological role: PBP2a is involved in bacterial cell wall synthesis.This protein confers resistance to β-lactam antibiotics by preventing these antibiotics from binding to penicillin-binding proteins, allowing the bacteria to continue synthesizing its cell wall despite the presence of the antibiotic.
• Biological role: This protein is involved in the formation of the endospore coat, a resistant structure that protects bacterial spores under extreme environmental conditions.Inhibiting this protein can disrupt spore formation, reducing bacterial survival in adverse conditions.
Three-dimensional crystal structures of target proteins were extracted from the Protein Data Bank (PDB).These structures were prepared by removing water molecules, ligands, and non-protein elements.The software tools Discovery Studio, Autodock 4 and Autodock Tools were used to further investigate compound-protein interactions and evaluate interaction energies using a three-dimensional grid [54].The dimensions of the central analysis grid were carefully adjusted for each protein to ensure accurate placement of ligands in the complex [44,53].The results of the study were visualized in two dimensions to better understand the binding interactions.

Molecular Dynamics (MD)
Molecular dynamics simulations used NAMD software [55] associated with CHARMM36 force field [56].The simulation setup consisted of enclosing the system in a cubic box, measuring 10 Å on each side, filled with TIP3P water molecules.The system was neutralized by adding NaCl ions at a concentration of 0.154 M, and applying the Monte Carlo technique [57].The initial phase involved minimizing the energy of each system using a 10,000-step gradient descent method.This was followed by a 100 ns stabilization phase under the NVT ensemble (number of particles, volume, temperature), keeping the system at a constant temperature of 310 K.This was followed by an additional 100 ns of unrestricted molecular dynamics simulation under the NPT (number of atoms, pressure, temperature) ensemble for each system [48].The stability of the systems was evaluated by analyzing the molecular dynamics trajectories through the Visual Molecular Dynamics (VMD) software [58].

In Silico Pharmacokinetics ADMET
The integration of computer technology has transformed the way we discover new drugs, making the process both faster and more accurate [58].Computational studies give us an improved understanding of a compound's behavior with regard to absorption, distribution, metabolism, excretion, and toxicity (ADMET).These analyses exploit information on how a drug acts to anticipate its effects from the earliest stages of development.The pkCSM online platform is particularly useful for assessing a drug's capacity to be absorbed by the human intestine, its distribution in the body, its biological transformation and elimination, as well as its toxicity profile [56].Computer simulation has thus become indispensable in the evaluation of pharmacokinetic parameters linked to ADMET.

Figure 2 .
Figure 2. Perspective view of isoxazole 4c with labeling scheme and 50% probability e
Figure 3.A portion of one ribbon viewed along the b-axis direction with C-H•••O depicted by dashed lines.Non-interacting hydrogen atoms are omitted for clarity

Figure 3 .
Figure 3.A portion of one ribbon viewed along the b-axis direction with C-H•••O hydrogen bonds depicted by dashed lines.Non-interacting hydrogen atoms are omitted for clarity (isoxazole 4c).

Figure 3 .
Figure 3.A portion of one ribbon viewed along the b-axis direction with C-H•••O hydrogen depicted by dashed lines.Non-interacting hydrogen atoms are omitted for clarity (isoxazole

Figure 4 .
Figure 4. Packing viewed along the a-axis direction with C-H•••O hydrogen bonds an H•••π(ring) interactions shown, respectively, by black and light blue dashed lines.Non-inte hydrogen atoms are omitted for clarity (isoxazole 4c).

Figure 5 .
Figure 5. Perspective view of isoxazole 4f with labeling scheme and 50% probability ellipsoid intramolecular C-H•••π(ring) interaction is depicted by a dashed line.

Figure 4 .
Figure 4. Packing viewed along the a-axis direction with C-H•••O hydrogen bonds and C-H•••π(ring) interactions shown, respectively, by black and light blue dashed lines.Non-interacting hydrogen atoms are omitted for clarity (isoxazole 4c).

Figure 3 .Figure 4 .
Figure 3.A portion of one ribbon viewed along the b-axis direction with C-H•••O hydrogen bon depicted by dashed lines.Non-interacting hydrogen atoms are omitted for clarity (isoxazole 4c)

Figure 5 .
Figure 5. Perspective view of isoxazole 4f with labeling scheme and 50% probability ellipsoids.T intramolecular C-H•••π(ring) interaction is depicted by a dashed line.

Figure 5 .
Figure 5. Perspective view of isoxazole 4f with labeling scheme and 50% probability ellipsoids.The intramolecular C-H•••π(ring) interaction is depicted by a dashed line.The isoxazole ring appears planar although the slight elongation of the displacement ellipsoids for O1 and N1 normal to the plane could suggest the presence of two opposite non-planar conformers.The mean planes of the C18•••C23 and the C24•••C29 rings are inclined to that of the isoxazole ring by 59.33(6) and 40.92(8) • , respectively, while the dihedral angles between the mean planes of the C5•••C10 and the C12•••C17 rings and

Figure 6 .
Figure 6.A portion of one ribbon viewed along the a-axis direction with C-H•••π(ring) interact depicted by dashed lines.Non-interacting hydrogen atoms and the intramolecular C-H•••π(r interactions are omitted for clarity (isoxazole 4f).

Figure 6 .
Figure 6.A portion of one ribbon viewed along the a-axis direction with C-H•••π(ring) interactions depicted by dashed lines.Non-interacting hydrogen atoms and the intramolecular C-H•••π(ring) interactions are omitted for clarity (isoxazole 4f).

Figure 6 .
Figure 6.A portion of one ribbon viewed along the a-axis direction with C-H•••π(ring) interactions depicted by dashed lines.Non-interacting hydrogen atoms and the intramolecular C-H•••π(ring) interactions are omitted for clarity (isoxazole 4f).

Table 1 . Physicochemical characteristics and characterization data of 4(a-f).
Scheme 2. Synthetic route of functionalized isoxazole compounds.Table 1. Physicochemical characteristics and characterization data of 4(a-f).

Table 4 .
In vitro antibacterial activity of 4(a-f).
a Results are expressed as means ± standard deviations of duplicate experiments; NT: not tested.-: no activity.

Table 4 .
In vitro antibacterial activity of 4

(a-f). Zone of Inhibition (ZI) in mm a
a Results are expressed as means ± standard deviations of duplicate experiments; NT: not tested.-: no activity.

Table 6 .
In silico ADMET predictions for